Compositions of hyaluronan with high elasticity and uses thereof

ABSTRACT

The present invention provides compositions comprising hyaluronan with high elasticity, as well as methods for improving joint function, reducing pain associated with joint function and treating osteoarthritis by introducing into a joint a therapeutically effective amount of a composition comprising hyaluronan with high elasticity.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/844,645, filed on Jul. 10, 2013, the entire contents of which arehereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Hyaluronan (HA) is a high average molecular weight linear polysaccharidewhich is found primarily in the extracellular matrix and pericellularmatrix, but has also been shown to occur intracellularly. The biologicalfunctions of HA include maintenance of the elastoviscosity of liquidconnective tissues such as joint synovial fluid and eye vitreous,control of tissue hydration and water transport, supramolecular assemblyof proteoglycans in the extracellular matrix, and numerousreceptor-mediated roles in cell detachment, mitosis, migration and tumordevelopment.

The analgesic effect of HA solutions on joint pain receptors is welldocumented. It has been previously suggested that HA of high averagemolecular weight, when injected intra-articularly, may be the mosteffective at reducing joint pain. There is experimental evidence tosuggest that high average molecular weight hyaluronan acts as anelastoviscous filter, buffering the transmission of mechanical forces toion channels responsible for the detection of injurious stimuli in jointpain nerve endings, thereby decreasing their excitation. However, thetype of interaction between HA molecules and ion channels leading to achange in the excitability of pain nerve endings is unknown. Also, therheological properties of HA solutions that are most appropriate tomaximize their analgesic effects on joint pain receptors have not beenfully investigated.

Pain associated with joint function, including pain associated with kneejoint function and osteoarthritis, can be treated by injection ofcompositions that include hyaluronan into the painful joint. However,current treatment methods do not result in a complete suppression of thepain or uniform reduction of pain in all patients. Hyaluronan-basedcompositions with improved properties are, therefore, needed in the art.

SUMMARY OF THE INVENTION

The present inventors have discovered that compositions comprising highconcentrations of HA, e.g., compositions having HA concentrations ofabout 30 mg/mL (about 3% weight/volume), or greater are surprisinglyeffective at treating joint pain. In fact, such HA compositions aresignificantly more effective at treating joint pain than Synvisc®, themost successful HA commercial product currently used forviscosupplementation. Without wishing to be bound by a specific theory,it is believed that the effectiveness of the HA compositions of theinvention comprising high concentrations of HA at treating joint pain isdetermined by their high elasticity, as is evidenced by the high valueof the elastic modulus G′. It is also believed, without wishing to bebound by a specific theory, that the effectiveness of the HAcompositions of the invention is determined by a relatively highprobability of interaction of HA molecules with pain transducingchannels, such as TRPV1, thereby reducing nociceptor excitability.

Accordingly, the present invention provides compositions comprisinghyaluronan, wherein the hyaluronan is present in the composition at aconcentration of greater than about 30 mg/mL; the hyaluronan has anaverage molecular weight of between about 1 and about 2 million; thehyaluronan is not cross-linked and/or is substantially free of chemicalmodifications; and wherein the composition is substantially free ofother pharmaceutically active substances.

In one embodiment, the other pharmaceutically active substance is aprotein. In another embodiment, the other pharmaceutically activesubstance is a glycosaminoglycan that is different from hyaluronan. Inyet another embodiment, the other pharmaceutically active substance ishydroxypropyl methyl cellulose. In a further embodiment, the otherpharmaceutically active substance is a local anesthetic, e.g., lidocaineor bupivacaine.

In some embodiments, hyaluronan is present in the composition at aconcentration of about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about55 mg/mL or about 60 mg/mL.

In one embodiment of the invention, the composition has an elasticity ofat least about 200 Pascal when measured at a frequency of 0.5 Hz, or anelasticity of at least about 1,000 Pascal when measured at a frequencyof 0.5 Hz. In another aspect, the composition has an elasticity of atleast about 2,000 Pascal when measured at a frequency of 0.5 Hz, or anelasticity of at least about 4,000 Pascal when measured at a frequencyof 0.5 Hz.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising hyaluronan, wherein the hyaluronan is present inthe composition at a concentration of about 40 mg/mL; the hyaluronan hasan average molecular weight of between about 1 and about 2 million; thehyaluronan is not cross-linked and/or is substantially free of chemicalmodifications; and wherein the composition is substantially free ofother pharmaceutically active substances.

In one embodiment, the other pharmaceutically active substance is aprotein. In another embodiment, the other pharmaceutically activesubstance is a glycosaminoglycan that is different from hyaluronan. Inyet another embodiment, the other pharmaceutically active substance ishydroxypropyl methyl cellulose. In a further embodiment, the otherpharmaceutically active substance is a local anesthetic, e.g., lidocaineor bupivacaine.

In one embodiment of the invention, the composition has an elasticity ofat least about 200 Pascal when measured at a frequency of 0.5 Hz, or anelasticity of at least about 1,000 Pascal when measured at a frequencyof 0.5 Hz. In another aspect, the composition has an elasticity of atleast about 2,000 Pascal when measured at a frequency of 0.5 Hz, or anelasticity of at least about 4,000 Pascal when measured at a frequencyof 0.5 Hz.

In one embodiment, the compositions of the invention are sterile. Inanother embodiment, the compositions are suitable for injection into asubject's joint, e.g., the knee, elbow, hip or other appendicular oraxial joints.

The present invention also provides methods of reducing at least onesymptom associated with joint dysfunction. The methods compriseadministering to a subject in need thereof a therapeutically effectiveamount of a composition of the invention, thereby reducing the at leastone symptom associated with joint dysfunction.

In some embodiments, the joint dysfunction is joint pain or jointdysfunction associated with osteoarthritis, post-arthroscopy,post-orthoplasty, post-injury or prolonged immobilization. In a specificembodiment, the joint dysfunction is joint pain.

In some embodiments, the administering of the composition of theinvention causes a reduction, e.g., at least a two-fold, at least athree-fold, at least a four-fold or at least a five-fold reduction, inthe number of nerve impulses induced by normal and abnormal movements inan experimental rat model of osteoarthritis.

In some embodiments, the administering of the HA composition of theinvention causes a reduction, e.g., at least a two-fold, at least athree-fold, at least a four-fold, at least a five-fold, at least asix-fold, at least a seven-fold or at least an eight-fold reduction, inthe number of nerve impulses induced by normal movements in anexperimental rat model of osteoarthritis.

In some embodiments, the administering of the HA composition causes areduction, e.g., at least a two-fold, at least a three-fold or at leasta four-fold reduction, in the number of nerve impulses induced byabnormal movements in an experimental rat model of osteoarthritis. In aspecific embodiment, the reduction is greater than the reduction causedby administering a comparable amount of Synvisc®.

In some embodiments, the HA composition is administered byintra-articular injection.

In another aspect, the present invention provides a pre-filled syringe,e.g., a sterile syringe, comprising the HA composition of the invention,as well as kits comprising such pre-filled syringes.

The present invention is further illustrated by the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, Panel A is a graph showing G′ values (in Pascal) as a functionof frequency (in Hz) for commercially available HA compositions used forviscosupplementation.

FIG. 1, Panel B is a graph showing G′ values (in Pascal) as a functionof frequency (in Hz) for HA compositions of the invention (Elastovisc™).This Figure demonstrates that the compositions of the invention arecharacterized by much higher G′ values than any of the tested HAcommercial products.

FIG. 2, Panel A is a graph showing the number of nerve impulses permovement in inflamed joints as a function of time after injection ofSynvisc® and various HA compositions of the invention.

FIG. 2, Panel B is a graph showing the mean number of nerve impulses permovement in inflamed joints as a function of molecular weight for 4% HAcompositions of the invention.

FIG. 2, Panel C is a graph showing the mean number of nerve impulses permovement in inflamed joints as a function of HA concentration.

FIG. 2, Panel D is a bar graph showing the mean number of nerve impulsesper movement in inflamed joints for various tested HA compositions.

FIG. 3, is a graph showing the number of nerve impulses evoked by thenon-noxious component of the joint movement cycle as a function of timemeasured in inflamed rat joints following injection with salinesolution, Synvisc® or HA composition of the invention (Elastovisc™, 4%HA, average molecular weight 1-2 million).

FIG. 4 is a graph showing the number of nerve impulses evoked by thenoxious component of the movement cycle as a function of time measuredin inflamed rat joints following injection with saline solution,Synvisc® or HA composition of the invention (Elastovisc™, 4% HA, averagemolecular weight 1-2 million).

FIG. 5, Panel A shows the average total number of impulses per movementrecorded at different times after the onset of inflammation followed byan intra-articular injection of saline or a 4% HA composition of theinvention.

FIG. 5, Panel B shows the percent difference in the mean total number ofimpulses per movement measured after saline or 4% HA injection, atdifferent times following injection.

FIG. 6, Panel A is a graph showing the mean number of impulses permovement with repeated movements in three un-injected intact joints(open circles) and in seven intact joints (filled circles) following anintra-articular injection of 4% HA composition of the invention 24 hoursearlier.

FIG. 6, Panel B is a bar graph showing the mean values ofmovement-evoked impulses per movement measured in un-injected intactjoints and in intact joints injected with a 4% HA composition of theinvention 24 hours earlier.

FIG. 6, Panel C is a graph showing the time course of the decrease inmovement-evoked activity in two different joint nociceptor fibers ofdifferent rats following intra-articular injection of a 4% HAcomposition into an intact knee joint.

FIG. 7 is a graph showing the number of movement-evoked nerve impulsesas a function of time measured in intact rat joints followingintra-articular injection with saline or three different volumes of theHA composition of the invention (Elastovisc™).

FIG. 8, Panels A1 and A2 show intracellular calcium rises evoked inHEK-TRPV1-EYFP(+) cells by heat after perfusion with saline (A1) or 400μg/mL HA (A2). Panel A3 shows the ratio of average amplitude changebetween responses evoked by successive heat pulses in control salinesolution and during perfusion with HA for HEK-TRPV1-EYFP(+) cells.

FIG. 8, Panels B1 and B2 show intracellular calcium rises evoked in DRGadult neurons by heat after perfusion with saline (B1) or 400 μg/mL HA(B2). Panel B3 shows the ratio of average amplitude change betweenresponses evoked by successive heat pulses in control saline solutionand during perfusion with 400 μg/mL HA for DRG adult neurons.

FIG. 8, Panels C1 and C2 show intracellular calcium rise in aHEK-TRPV1-EYFP (+) cell in response to 100 nM capsaicin and 10 μMCarbachol in control solution (C1) and under exposure to 400 μg/mL HA(C2). Panel C3 shows the average amplitude of the response to capsaicin(filled bars) and Carbachol (striped bars) in HEK-TRPV1-EYFP (+) cellsduring perfusion with control saline solution and in the presence of HA.

FIG. 8, Panels D1 and D2 show intracellular calcium responses of DRGadult sensory neurons to 100 nM capsaicin and 30 mM KCl during perfusionwith saline (D1) and with 400 μg/mL HA (D2). Panel D3 shows the averageamplitude of the intracellular calcium responses to capsaicin (filledbars) or KCl (striped bars) in control saline solution and in thepresence of HA.

FIG. 9, Panel A shows I-V relationships of capsaicin-evoked currents inHEK-293-TRPV1 cells in saline solution (black trace) and in the 400μg/ml HA solution (gray trace).

FIG. 9, Panel B shows the average current measured at +80 mV potential,obtained from I-V curves shown in Panel A.

FIG. 9, Panel C shows values of different parameters measured from theramps, fitted with a function that combines a linear conductancemultiplied by a Boltzmann activation term,I=g×(V−E_(rev))/(1+exp((V_(1/2)−V/S)).

FIG. 9, Panel D shows the whole-cell currents measured at −60 mVpotential, in response to 1 μM capsaicin, in control conditions (toptrace), and in cells pre-incubated for 30 minutes and continuouslyperfused with HA (bottom trace).

FIG. 9, Panel E shows average values of peak currents in HEK-293-TRPV1cells evoked by capsaicin at −60 mV in saline and in the presence of HA.

FIG. 10, Panel A shows a sample record of TRPV1 single channel activityunder perfusion with saline solution.

FIG. 10, Panel B shows a sample recording of single-channel activityfrom cells incubated in HA and recorded under perfusion with HAsolution. The insets represent single channel amplitude probabilityhistograms of each recording.

FIG. 10, Panel C shows I-V curves obtained in control conditions (blacksquares) and after exposure to HA (gray circles).

FIG. 10, Panel D shows single channel amplitudes obtained fromindividual patches, represented as squares (control) and circles(HA-treated).

FIG. 10, Panel E shows the probability of the open state for differentpatches. Larger symbols represent the data from the measurementsperformed in the traces shown in Panels A and B.

FIG. 11, Panel A shows a sample record of the response to capsaicin in asingle DRG neuron perfused with saline solution.

FIG. 11, Panel B shows a sample record of capsaicin stimulation in a DRGneuron treated with HA and recorded in the presence of HA.

FIG. 11, Panel C shows a sample record in a DRG neuron treated with HA,in which the response to capsaicin was abrogated while a normal responseto KCl was still evoked.

FIG. 11, Panel D shows the mean firing frequency (left) and individualdata (right) of DRG neurons under control conditions and after exposureto HA.

FIG. 12, shows the latency of the nociceptive response of mice in thehot plate test to a plate temperature of 52° C. in wild type (leftpanel) and in TRPV1 knock-out mice (right panel), after subcutaneousinjection in the paw of 10 μl of sterile saline, HA, hyaluronidase(Hyasa 6U) or hyaluronidase followed by another injection of HA.

FIG. 13, Panel A shows the nerve impulse activity evoked by a jointmovement in an anesthetized rat.

FIG. 13, Panel B is an expanded trace for one of the nerve fibersactivated by the joint movement. Panel B shows that this nerve fiber isrecruited by a rapid, bolus injection of capsaicin into the joint artery(upper trace). Following an intra-articular injection of HA, theresponse to capsaicin decreases gradually over time (medium trace, 180min; lower trace, 240 min).

FIG. 13, Panel C is a graph showing the mean values of the firingresponse to intra-arterial capsaicin, measured in animals that receivedan injection of intra-articular saline (filled squares) or HA (opensquares).

FIG. 14 shows the pressure required to eject a 4% HA solution throughneedles of different sizes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions comprising highconcentrations of hyaluronan (HA). Such compositions were determined tohave high elasticity, e.g., high elastic modulus G′, when measured atfrequencies of 0.1-10 Hz. It has been presently discovered that HAcompositions characterized by high elasticity, e.g., compositionscomprising high concentrations of HA, are surprisingly effective attreating joint pain, such as joint pain caused by osteoarthritis. Theaverage molecular weight of HA comprised in the compositions of theinvention may be 2 million or less, e.g., between about 1-2 million.

The above discovery was unexpected because it was previously believedthat the effectiveness of HA compositions in treating joint pain wasdependent on the average molecular weight of HA, and not on itsconcentration. In particular, it was believed that high averagemolecular weight HA compositions, e.g., compositions comprising HAhaving an average molecular weight of greater than 2 million, were themost effective for treating joint pain.

Hyaluronan (also called hyaluronic acid or hyaluronate or HA) is aglycosaminoglycan present in joint fluid and in the synovial tissuearound it. This highly elastoviscous, polydisperse polysaccharide is amajor component of the synovial fluid that fills the intercellular spacein the synovium, the connective tissue that surrounds the joint space.

The elastoviscous properties of synovial fluid are critical to itsphysiological functions. Synovial fluid must be capable of viscous flowto participate in the fluid movements that are critical forintra-articular metabolism. However, the same synovial fluid must alsobehave as a shock absorber, using its elastic properties to store theimpact of mechanical stress on the joint in a way that limits thedeformation of tissue surfaces, fibrous structures, nerve endings andcells.

The ability of HA contained in the synovial fluid and tissues tomodulate the response of a joint to different types of movements isexplained by the fact that HA possesses both viscous and elasticproperties. During slow movement, the synovial fluid that fills thetissue is exposed to low deformation frequencies and the rate at whichenergy is transmitted to the hyaluronan network is low enough to allowtime for the hyaluronan molecules to adjust their configuration and lineup in the direction of flow. Thus, during slow movement, the energyapplied to the synovial fluid is predominantly dissipated as viscousflow and heat. This alignment of hyaluronan molecules is alsoresponsible for the pseudoplasticity of synovial fluid (shear thinning,or the decrease in viscosity with increasing flow rate). However, whenthe hyaluronan network in synovial fluid is subjected to a high rate ofdeformation, such as during running or jumping, the stress istransferred rapidly and so the hyaluronan molecules have insufficienttime to adjust their configuration. Instead, HA acts as coiled molecularshock absorber, storing the energy transmitted as elastic deformation,and behaving like an elastic solid.

The strain frequency of the applied mechanical stress determines whetherthe hyaluronan acts predominantly as a viscous fluid or an elastic shockabsorber. Two values are used to describe the rheological profile of anelastoviscous fluid: the elasticity modulus, G′, is a measure ofelasticity, while the viscosity modulus, G″, is a measure of viscosity.When HA is subjected to a high rate of deformation (e.g., during runningor jumping), the HA molecules act as a coiled molecular shock absorberand behave like an elastic solid. When HA is subjected to a low rate ofdeformation (e.g., during slow movement), the energy applied to thesynovial fluid is dissipated as viscous flow and heat.

In the 1960s, it was discovered that hyaluronan, when injected intoinflamed knee joints, reduces pain. As a result of this discovery,highly purified HA solutions were developed for the treatment ofarthritic pain in humans and animals. This new therapeutic use wascalled viscosupplementation. HA can act as an elastoviscous cover,reducing the mechanical force reaching the ion channels responsible forthe activation of the pain nerve fibers by noxious stimuli, therebyreducing the opening probability of certain channels, decreasing thenumber of nerve impulses in pain terminals and, thus, reducing painsensations. It was also shown that an increase of protection for thepain receptors improves the healing processes in the joint.

The average molecular weight of hyaluronan in human synovial fluid of ahealthy individual is between 3-4 million, while the average molecularweight of hyaluronan in the pathological joint is between 0.5 to 2million. It was thought that hyaluronan used in the treatment of jointpain, e.g., joint paint associated with osteoarthritis, had to have asimilar or even higher average molecular weight than the hyaluronanpresent in the healthy joint. In order to reduce pain and inflammation ahyaluronan of high average molecular weight was used that was the samesize or larger as present in the joint prior to the joint becomingpathological (painful).

In the synovial fluid of healthy human knee joints, hyaluronan is foundat an average concentration of around 321 mg/100 mL (range of250-368/mg/100 mL). In pathological joints, the synovial fluid has beenfound to have a lower concentration, mostly between 40-188 mg per 100mL. It was also thought that hyaluronan used in the treatment of jointpain, e.g., in joint pain associated with osteoarthritis, had to beadministered at the same or even a higher concentration as thehyaluronan present in the healthy joint. Commercially availablehyaluronan compositions for injection into a painful or otherwisepathological joint have a concentration of up to 2.2% (22 mg/mL). Thus,the concentration of hyaluronan in compositions available for injectioninto the joint is much greater than the concentration of hyaluronan inhealthy joints.

There are several hyaluronan products on the market worldwide for theimprovement of joint function and the treatment of pain associated withjoint function (see e.g., Table 1 in Example 1). These productsgenerally have a high average molecular weight of hyaluronan and arelatively low concentration (1-2.2%). As noted above, hyaluronan inhuman synovial fluid of a healthy individual is between 3-4 millionaverage molecular weight. It was thought that hyaluronan with a highaverage molecular weight would be the most effective in the treatment ofjoint pain.

The ultrapure hyaluronan product Synvisc® (described, for instance, byEndre A. Balazs in U.S. Pat. No. 4,141,973), is the hyaluronan productwith the highest average molecular weight available forviscosupplementation (5 million), was found to be the most active “painkiller” in pathological animal and human joints. Synvisc® also contains20% of a cross-linked, highly hydrated hyaluronan gel. Synvisc® reducesthe impulse activity in sensory fibers evoked by movements in normal andinflamed joints, thereby reducing the pain associated with jointfunction. Synvisc® is thought to be the most effective of the currentlyavailable commercial HA products in the treatment of pain. However, theproduction of sterile hyaluronan with high average molecular weight ischallenging, and this is believed to be a reason that all commerciallyavailable hyaluronan products except Synvisc® have a significantly loweraverage molecular weight.

Many hyaluronan products currently on the market have a concentration ofhyaluronan of about 0.8% (Synvisc®), 1.0% (Euflexxa®, Supartz®,Hyalgan®, Gel-One®, Synocrom®, Synocrom® Mini), 1.5% (Orthovisc®), 2%(Synocrom® Forte, Synocrom® Forte One, Synolis V-A) or 2.2% (Monovisc®).These hyaluronan products have a percentage of hyaluronan not higherthan 0.8-2.2%, because such a concentration is already much higher thanthe concentration in healthy tissue. It was assumed in the art thatconcentrations even higher than 0.8-2.2% would be too high compared tothe hyaluronan concentration in the healthy human joint.

It has now been recognized for the first time that inflamed and painfuljoints are more responsive to HA compositions of high elasticity than toHA compositions with low elasticity. Accordingly, the present inventionprovides compositions of hyaluronan with high elastic properties andshows the superior properties in the treatment of joint pain ofcompositions of hyaluronan with high elastic properties (e.g.,Elastovisc™) as compared to currently available compositions ofhyaluronan with low elasticity. The compositions of hyaluronan with highelastic properties disclosed herein (e.g., Elastovisc™) are based on thediscovery that the anti-pain effect of hyaluronan in the joint does notdepend on the viscosity of the product, but on its elasticity.Consequently, the therapeutic success of the compositions of hyaluronandisclosed herein depends on the elasticity of the hyaluronancompositions used. While not being limited to a particular mechanism, itis thought that the high elasticity hyaluronan concentrations providedherein, persist in the joint for a long time (e.g., weeks or longer),and influence not only the pain receptors, but interact with and/orremove pain-causing chemical agents.

As disclosed herein, in one embodiment, compositions of hyaluronan withhigh elasticity can be generated by increasing the concentration of thehyaluronan in the compositions. It is unexpectedly shown herein thatcompositions of hyaluronan with a high concentration of hyaluronan, evenwith an average molecular weight of 1-2 million, can be used in thetreatment of joint pain. This result is unexpected because prior to theinstant invention it was thought that only high average molecular weightcompositions of hyaluronan (e.g., Synvisc®) could have a strong painreducing effect. The result is further unexpected because prior to theinstant invention it was thought that concentrations greater than0.8-2.2% (which are already much higher than the concentrations found inthe healthy joint), would have no additional beneficial effects or couldeven be too high in comparison with the concentration in the healthyjoint. Additionally, the present inventors have discovered that the highconcentration of HA molecules facilitates their interaction with TRPV1channels of nociceptors, thereby reducing the responsiveness of thenociceptors to noxious stimuli.

I. Hyaluronan Compositions of the Invention

The present invention provides pharmaceutical compositions comprisinghyaluronan (HA). In some embodiments, a composition of the inventioncomprises hyaluronan, wherein the hyaluronan is present in thecomposition at a concentration of greater than about 30 mg/mL (orgreater than about 3% weight/volume); the hyaluronan has an averagemolecular weight of between about 1 and about 2 million; the hyaluronanis not cross-linked and/or is substantially free of chemicalmodifications; and wherein the composition is substantially free ofother pharmaceutically active substances.

For example, the HA concentration in the compositions of the inventionmay be about 30 mg/mL (or about 3% w/v), about 35 mg/mL (or about 3.5%w/v), about 40 mg/mL (or about 4% w/v), about 45 mg/mL (or about 4.5%w/v), about 50 mg/mL (or about 5% w/v), about 55 mg/mL (or about 5.5%w/v), about 60 mg/mL (or about 6% w/v), about 65 mg/mL (or about 6.5%w/v), about 70 mg/mL (or about 7% w/v), about 75 mg/mL (or about 7.5%w/v), about 80 mg/mL (or about 8% w/v), about 85 mg/mL (or about 8.5%w/v), about 90 mg/mL (or about 9% w/v), about 95 mg/mL (or about 9.5%w/v), about 100 mg/mL (or about 10% w/v), about 105 mg/mL (or about10.5% w/v), about 110 mg/mL (or about 11% w/v) about 115 mg/mL (or about11.5% w/v), about 120 mg/mL (or about 12% w/v), about 125 mg/mL (orabout 12.5% w/v), about 130 mg/mL (or about 13% w/v), about 135 mg/mL(or about 13.5% w/v), about 140 mg/mL (or about 14% w/v), about 145mg/mL (or about 14.5% w/v), or about 150 mg/mL (or about 15% w/v). In aspecific embodiment, the HA is present in the composition at aconcentration of about 40 mg/mL (or about 4% w/v). In another specificembodiment, the HA is present in the composition of the invention at theconcentration of about 60 mg/mL (or about 6% w/v).

In certain embodiments, the hyaluronan used in the compositions of theinvention is not cross-linked and/or is free of chemical modifications.For example, the hyaluronan used in the compositions of the invention isfree from amidation that may be formed by a reaction between thecarboxyl group of HA and the amine group of a derivatizing agent asdescribed, e.g., in EP Patent No. 1095064 B1. The hyaluronan used in thecompositions of the invention may also be free from chemicalmodifications and/or cross-links that may result from the reaction ofhyaluronan with a carbodiimide, such as a monocarbodiimide or abiscarbodiimide, as described, for example, in U.S. Pat. No. 8,323,617.

In some embodiments, the HA compositions of the invention are free fromother pharmaceutically active substances. As used herein, a“pharmaceutically active substance” is a substance that is capable ofexerting a biological effect on a subject, e.g., a human or an animalsubject. This term “pharmaceutically active substance” also comprisessubstances that can modulate the biological effect of an HA compositionwhen the composition is administered to a subject, e.g., modulate theability of the HA composition to reduce pain in an inflamed joint. Incertain embodiments, the pharmaceutically active substance is a protein,e.g., a bone morphogenic protein (BMP), such as rhGDF-5. In certainembodiments, the pharmaceutically active substance is aglycosaminoglycan (GAG) that is different from HA, e.g., chondroitin. Insome embodiments, the pharmaceutically active substance is hydroxypropylmethyl cellulose. In other embodiments, the pharmaceutically activesubstance is a topical anesthetic, such as a lidocaine or a bupivacaine.

In certain embodiments, the HA compositions of the invention are freefrom molecules capable of scavenging free radicals, such as sorbitol. Inother embodiments, the HA compositions of the invention are free frommolecules that diminish the elasticity of HA, for example, dextran orsucrose.

In some embodiments, an HA composition of the invention consistsessentially of HA present at a concentration of greater than about 30mg/mL (about 3% w/v), or about 40 mg/mL (about 4% w/v) in aphysiological buffer, e.g., a phosphate buffer or a bicarbonate buffer,and having the average molecular weight of between about 1 million andabout 2 million. In a specific embodiment, an HA composition of theinvention consists essentially of HA present at a concentration of about40 mg/mL (or about 4% w/v), and having the average molecular weight ofbetween about 1 million and about 2 million.

In another embodiment, the HA compositions of the invention may beadministered to a subject in need thereof via an injection using aninjection device, such as a needle, a trocar, a cannula or a perfusiondevice. The injection device suitable for injecting the HA compositionsof the invention may have a nominal diameter of 2.11 mm or greater(corresponding to 14 G needle, or a needle gauge of 14 of greater). Insome embodiments, the HA compositions of the invention may be tooviscous for administration using smaller needles, e.g., needles having anominal diameter of less than 2.11 mm. In other embodiments, the HAcompositions of the invention may allow administration using smallerinjection devices having a nominal diameter of less than 2.11 mm.

For example, a device suitable for injecting the HA compositions of theinvention, such as a syringe, may have a nominal diameter of about 0.31mm, 0.34 mm, 0.36 mm, 0.41 mm, 0.474 mm, 0.46 mm, 0.49 mm, 0.515 mm,0.51 mm, 0.54 mm, 0.57 mm, 0.59 mm, 0.642 mm, 0.64 mm, 0.67 mm, 0.718mm, 0.72 mm, 0.77 mm, 0.82 mm, 0.87 mm, 0.91 mm, about 0.99 mm, about1.07 mm, about 1.17 mm, about 1.27 mm, about 1.42 mm, about 1.47 mm,about 1.57 mm, about 1.65 mm, about 1.73 mm, about 1.83 mm, about 1.98mm, about 2.11 mm, about 2.26 mm, about 2.41 mm, about 2.54 mm or about2.77 mm, corresponding, respectively, to gauge of 30, 29, 28, 27, 26 s,26, 25.5, 25 s, 25, 24.5, 24, 23.5, 23 s, 23, 22.5, 22 s, 22, 21.5, 21,20.5, 20, 19.5, 19, 18.5, 18, 17.5, 17, 16.5, 16, 15.5, 15, 14.5, 14,13.5, 13, 12.5 or 12 (or 30 G, 29 G, 28 G, 27 G, 26 sG, 26 G, 25.5 G, 25sG, 25 G, 24.5 G, 24 G, 23.5 G, 23 sG, 23 G, 22.5 G, 22 sG, 22 G, 21.5G, 21 G, 20.5 G, 20 G, 19.5 G, 19 G, 18.5 G, 18 G, 17.5 G, 17 G, 16.5 G,16 G, 15.5 G, 15 G, 14.5 G, 14 G, 13.5 G, 13 G, 12.5 G or 12 G needles).In one embodiment, the HA compositions of the invention may beadministered using an 18 G syringe needle having a nominal diameter ofabout 1.27 mm.

In some embodiments, the HA compositions of the invention may be tooviscous for administration using smaller needles, e.g., needles having anominal diameter of.

The hyaluronan in the compositions of the invention may have anelasticity of at least 100 Pascal when measured at a frequency of 0.5Hz, or an elasticity of at least 400 Pascal when measured at a frequencyof 0.5 Hz, or an elasticity of at least 1,000 Pascal when measured at afrequency of 0.5 Hz, or an elasticity of at least 2,000 Pascal whenmeasured at a frequency of 0.5 Hz, or an elasticity of at least 4,000Pascal when measured at a frequency of 0.5 Hz, or an elasticity ofbetween 400 and 5,000 Pascal when measured at a frequency of 0.5 Hz.

It should be appreciated that a variety of methods are available formeasuring the elasticity of a biopolymer such as hyaluronan. In oneembodiment, the present invention provides compositions comprisinghyaluronan with high elasticity, wherein the elasticity is measured aspressure (expressed in Pascal) at a specific frequency (expressed inHertz). In some embodiments, the frequencies used herein correspond to aparticular joint movement. For instance, the frequencies that may beused to evaluate the elasticity of the hyaluronan compositions providedherein, may be measured at 0.5 Hz (corresponding to walking), 2.5 Hz,(corresponding to running), or 5.0 Hz (corresponding to jumping). Thesefrequencies are applicable to the knee joint, but other joints that havea similar exposure (e.g., hip, ankles) may experience the same stressfrequency. Furthermore, while the elasticity may be expressed as apressure at a specific function associated with knee function (e.g.,walking, running), elasticity may also be expressed as pressure exertedby a non-walking movement (e.g., rotation of an elbow or shoulder or awrist movement).

It should further be appreciated that the elasticity may be expressed inany relevant frequency. Thus, for instance, in one embodiment, theelasticity is expressed based on a “running” frequency of 2.5 Hz and acomposition comprising hyaluronan with high elasticity is a compositionwith an elasticity of at least 200 Pa at a frequency of 2.5 Hz.Similarly, in one embodiment, the elasticity is expressed based on a“jumping” frequency of 5.0 Hz and a composition comprising hyaluronanwith high elasticity is a composition having an elasticity of at least400 Pa at a frequency of 5.0 Hz.

In one aspect, the present invention provides a composition comprisinghyaluronan, wherein the composition has an elasticity of at least 100Pascal when measured at a frequency of 0.5 Hz. In some embodiments, thecomposition has an elasticity of at least 300 Pascal when measured at afrequency of 2.5 Hz. In some embodiments, the composition has anelasticity of at least 350 Pascal when measured at a frequency of 5.0Hz.

In one aspect, the present invention provides a composition comprisinghyaluronan, wherein the composition has an elasticity of at least 400Pascal when measured at a frequency of 0.5 Hz. In some embodiments, thecomposition has an elasticity of at least 750 Pascal when measured at afrequency of 2.5 Hz. In some embodiments, the composition has anelasticity of at least 900 Pascal when measured at a frequency of 5.0Hz.

In one aspect, the present invention provides a composition comprisinghyaluronan, wherein the composition has an elasticity of at least 1000Pascal when measured at a frequency of 0.5 Hz. In some embodiments, thecomposition has an elasticity of at least 1600 Pascal when measured at afrequency of 2.5 Hz. In some embodiments, the composition has anelasticity of at least 2000 Pascal when measured at a frequency of 5.0Hz.

In one aspect, the present invention provides a composition comprisinghyaluronan, wherein the composition has an elasticity of at least 2600Pascal when measured at a frequency of 0.5 Hz. In some embodiments, thecomposition has an elasticity of at least 4000 Pascal when measured at afrequency of 2.5 Hz. In some embodiments, the composition has anelasticity of at least 4500 Pascal when measured at a frequency of 5.0Hz.

In one aspect, the present invention provides a composition comprisinghyaluronan, wherein the composition has an elasticity of at least 4000Pascal when measured at a frequency of 0.5 Hz. In some embodiments, thecomposition has an elasticity of at least 5000 Pascal when measured at afrequency of 2.5 Hz. In some embodiments, the composition has anelasticity of at least 6000 Pascal when measured at a frequency of 5.0Hz.

In some embodiments, the composition has an elasticity of between 100and 10,000 Pascal when measured at a frequency of 0.5 Hz. In someembodiments, the composition has an elasticity of between 400 and 5,000Pascal when measured at a frequency of 0.5 Hz. In some embodiments, thecomposition has an elasticity of between 1,000 and 2,000 Pascal whenmeasured at a frequency of 0.5 Hz.

In some embodiments, the composition has an elasticity of between 300and 10,000 Pascal when measured at a frequency of 2.5 Hz. In someembodiments, the composition has an elasticity of between 750 and 6,000Pascal when measured at a frequency of 2.5 Hz. In some embodiments, thecomposition has an elasticity of between 1,500 and 4,000 Pascal whenmeasured at a frequency of 2.5 Hz.

In some embodiments, the composition has an elasticity of between 300and 10,000 Pascal when measured at a frequency of 5.0 Hz. In someembodiments, the composition has an elasticity of between 900 and 7,000Pascal when measured at a frequency of 5.0 Hz. In some embodiments, thecomposition has an elasticity of between 2,000 and 5,000 Pascal whenmeasured at a frequency of 5.0 Hz.

In some embodiments, the elasticity is measured by using a suitabledevice (e.g., a rheometer). In some embodiments, the elasticity ismeasured by using a Stresstech High Resolution Research Rheometer(Reologica Instruments AB). In some embodiments, the elasticity isdetermined at ambient temperature and pressure. However, it should beappreciated that elasticity may also be measured at non-ambienttemperature and/or pressure. It should further be appreciated that aperson of ordinary skill in the art knows how to convert a magnitude ofelasticity determined at various temperatures and pressures into amagnitude of elasticity at ambient temperature and pressure.

As disclosed herein, high elasticity compositions of hyaluronan can beprepared by increasing the concentration of hyaluronan in thecomposition. Thus, in one aspect, the present invention providescompositions having high elasticity that comprise a high percentage ofhyaluronan. For example, the compositions of the invention comprise atleast 3.0% of hyaluronan (weight by volume), at least 3.5% of hyaluronan(weight by volume), at least 4.0% of hyaluronan (weight by volume), atleast 4.5% of hyaluronan (weight by volume), at least 5.0% of hyaluronan(weight by volume), at least 5.5% of hyaluronan (weight by volume), atleast 6.0% of hyaluronan (weight by volume), at least 6.5% of hyaluronan(weight by volume), at least 7.0% of hyaluronan (weight by volume), atleast 7.5% of hyaluronan (weight by volume), at least 8.0% of hyaluronan(weight by volume), at least 8.5% of hyaluronan (weight by volume), atleast 8.9% of hyaluronan (weight by volume), at least 9.0% of hyaluronan(weight by volume), at least 10.0% of hyaluronan (weight by volume), atleast 11.0% of hyaluronan (weight by volume), at least 12.0% ofhyaluronan (weight by volume), at least 13.0% of hyaluronan (weight byvolume), at least 14.0% of hyaluronan (weight by volume), or at least15.0%, or more, of hyaluronan (weight by volume).

Ranges intermediate to the recited values are also intended to be partof this invention. For example, hyaluronan content in the compositionsof the invention may be between about 3% and about 15% (weight/volume),between about 3% and about 10% (weight/volume), about 3.5% and about 9%(weight/volume), about 4% and about 8% (weight/volume), or about 5% andabout 7% (weight/volume).

It should further be appreciated that the amount of hyaluronan in aparticular volume may also be expressed by alternative means (e.g.,gram/liter or mol/liter). A person of ordinary skill in the art wouldknow how to convert the various means of expressing the amount ofhyaluronan in a particular volume.

As indicated above, it is unexpectedly shown herein that compositions ofhyaluronan with a high concentration of hyaluronan, even with an averagemolecular weight of about 1-2 million, are particularly effective in thetreatment of joint pain. Thus, in some embodiments of the compositionsof hyaluronan provided herein, the average molecular weight ofhyaluronan is less than 2 million, less than 1.9 million, less than 1.8million, less than 1.7 million, less than 1.6 million, less than 1.5million, less than 1.4 million, less than 1.3 million, less than 1.2million, less than 1.1 million, less than 1 million, less than 0.9million, less than 0.8 million, less than 0.7 million, less than 0.6million, or less than 0.5 million.

Ranges intermediate to the recited values are also intended to be partof this invention. For example, in the compositions of hyaluronanprovided herein, the average molecular weight of hyaluronan is between 1and 2 million, between 1 and 1.5 million, between 0.5 and 1 million,between 0.5 and 2 million, or between 0.9 and 1.4 million.

In some embodiments of the compositions of hyaluronan provided herein,the majority of the hyaluronan present in the composition falls withinthe average molecular weight range provided herein. Thus, for instance,in compositions with an average molecular weight of hyaluronan ofbetween 0.2 and 2 million, at least 95% of the hyaluronan present in thecomposition falls within the range of between 0.2 and 2 million. In someembodiments, at least 50% of the hyaluronan present in the compositionsprovided herein falls within the recited range of average molecularweight. In some embodiments, at least 60% of the hyaluronan present inthe compositions provided herein falls within the recited range ofaverage molecular weight. In some embodiments, at least 70% of thehyaluronan present in the compositions provided herein falls within therecited range of average molecular weight. In some embodiments, at least80% of the hyaluronan present in the compositions provided herein fallswithin the recited range of average molecular weight. In someembodiments, at least 90% of the hyaluronan present in the compositionsprovided herein falls within the recited range of average molecularweight. In some embodiments, at least 95% of the hyaluronan present inthe compositions provided herein falls within the recited range ofaverage molecular weight. In some embodiments, at least 98% of thehyaluronan present in the compositions provided herein falls within therecited range of average molecular weight. In some embodiments, at least99% of the hyaluronan present in the compositions provided herein fallswithin the recited range of average molecular weight. In someembodiments, at least 99.9% of the hyaluronan present in thecompositions provided herein falls within the recited range of averagemolecular weight.

II. Sources of Hyaluronan

The hyaluronan used in the compositions and methods provided herein maybe obtained from any source. In general, hyaluronan has the samechemical structure, regardless of its origin (e.g., chicken or roostercomb, human or bacterial cell wall). Hyaluronan can be obtained, forinstance, from chicken or rooster comb, from bacterial cell walls andfrom human tissue (vitreous of the eye, synovial fluid from the joints,etc.). In some embodiments, the hyaluronan is isolated from chickencombs. In some embodiments, the hyaluronan is isolated from human tissuee.g., umbilical cord, vitreous of the eye, synovial fluid from thejoints. In some embodiments, the hyaluronan is isolated from cellculture. In some embodiments, the hyaluronan is isolated from bacterialcell walls. The isolation of hyaluronan from various sources is known toa person of ordinary skill in the art. For instance, the harvest andpurification of hyaluronan from rooster combs is described in U.S. Pat.No. 4,141,973, while the harvest and purification of hyaluronan frombacterial sources is described in U.S. Pat. No. 4,517,295. In someembodiments, the hyaluronan is purified and harvested to a solution with0.15 M NaCl at a pH of 6-8. Generally, the hyaluronan obtained from thevarious sources will be free of proteins or glycosaminoglycans otherthan hyaluronan.

In some embodiments, the isolated hyaluronan is further purified toobtain hyaluronan with a desired average molecular weight range (e.g.,through column chromatography). Methods for purifying hyaluronan with adesired average molecular weight range are known to a person of ordinaryskill in the art.

In one aspect, the hyaluronan with high elasticity disclosed herein isunmodified hyaluronan. However, it should be appreciated that in someembodiments, the hyaluronan may be chemically modified. For instance,the hyaluronan may be chemically modified to increase the elasticity ofthe hyaluronan.

III. Sterilization of the Hyaluronan Compositions of the Invention

In some embodiments, the compositions of the invention are sterile. A“sterile composition”, as used herein, refers to a composition that issafe to be administered to a subject, e.g., a human subject. Thus, asterile composition will only have a minimal number of agents that cancause unwanted side effects such as an unwanted immune response, e.g.,inflammation or infection.

Methods for sterilizing compositions of hyaluronan are known in the artand include, for example, heat or steam sterilization, e.g., byautoclaving. In some embodiments, the HA compositions of the inventionare sterilized by heating the compositions. In some embodiments, the HAcompositions of the invention are sterilized by including the HAcomposition in a syringe and autoclaving the HA containing syringe at131° C. for 2 minutes or 121° C. for 15 minutes followed by immediatecooling.

IV. Additional Components for the Hyaluronan Compositions of theInvention

The HA compositions of the invention may include additional componentsthat may stabilize the hyaluronan and/or make the composition moresuitable for administration to a subject. In some embodiments, the HAcompositions of the invention may include a buffer. Buffers are added inorder to allow for a stable pH. Suitable buffers for use in the presentinvention include phosphate buffers and bicarbonate buffers. In someembodiments, the buffer is a tris-phosphate buffer. In some embodiments,the buffer is present in a concentration of between 1 mM and 100 mM,between 2 mM and 50 mM, or between 5 mM and 20 mM. In some embodiments,the buffer concentration is less than 1 mM. In some embodiments, thebuffer concentration is more than 100 mM. In some embodiments, thebuffer concentration is 10 mM. It should be appreciated that the bufferconcentration is dependent on the nature of the buffer that is beingused. In some embodiments, the pH of the composition is between pH 7 andpH 9 or between pH 7.5 and pH 8.5. In some embodiments, the pH of thecomposition is 8.0. In some embodiments, the pH of the composition is7.5. In some embodiments, the pH of the composition is 8.5. If needed,acid (such as HCL) or base (such as NaOH) can be added to thecomposition to attain the desired pH.

In some embodiments, the hyaluronan compositions include a buffer, e.g.,a physiologically compatible buffer, but do not include any additionalcomponents.

In some embodiments, the composition includes a stabilizing excipient,such as carboxylic acid or a salt thereof. In some embodiments, thecomposition includes a monocarboxylic acid and/or salt thereof. In someembodiments, the composition includes a gluconic acid and/or sodiumgluconate. In some embodiments, the composition includes a dicarboxylicacid and/or a salt thereof. In some embodiments, the compositionincludes a citric acid, succinic acid, malonic acid, maleic acid,tartaric acid and or a salt thereof. In some embodiments, the carboxylicacid is sodium citrate. In some embodiments, the composition includes atricarboxylic aid and/or a salt thereof. In some embodiments, thecomposition includes a nitrilotriacetic acid and/or sodiumnitrilotriacetic acid. In some embodiments, the composition includes atetracarboxylic acid and/or salt thereof. In some embodiments, thecomposition includes a ethylenediaminetetracetic acid (EDTA) and/orsodium EDTA. In some embodiments, the composition includes apentacarboxylic acid and/or a salt thereof. In some embodiments, thecomposition includes a diethylenetriaminepentaacetic (DTPA) acid and/orsodium DTPA. Suitable carboxylic acids include, but are not limited to,citrate compounds, such as sodium citrate; tartrate compounds, succinatecompounds, and EDTA. Kaushil et al. in Protein Science 1999 8: 222-233,and Busby et al. in the Journal of Biological Chemistry Volume 256,Number 23 pp 12140-1210-12147 describe carboxylic acids and their uses.In some embodiments, the stabilizing excipient has a concentration ofbetween 50 to 600 mM, between 250 to 500 mM, or between 250 to 350 mM.In some embodiments, the concentration of the stabilizing excipient is300 mM. In some embodiments, the concentration of the stabilizingexcipient is less than 100 mM. In some embodiments, the concentration ofthe stabilizing excipient is more than 600 mM.

In some embodiments, the HA compositions of the invention include asugar (e.g., a disaccharide sugar). Disaccharide sugars that can beadded to the composition include, but are not limited to, sucrose,lactulose, lactose, maltose, trehalose, cellobiose, dextrose anddextran. In some embodiments, the sugar is present at between 0.5 to 5%(wt/volume). In some embodiments, the sugar is present at between 1 to2% (wt/volume). In one embodiment, the sugar is present at 1%. In someembodiments, the sugar is present at less than 1% (wt/volume). In someembodiments, the sugar is present at more than 5% (wt/volume). In oneembodiment, the sugar is sucrose or trehalose and is present at 1%(wt/volume).

In some embodiments, the HA compositions of the invention include salts.Salts that can be used in the compositions include sodium chloride andother physiological compatible salts. In some embodiments, the saltconcentration is between 10 mM and 250 mM, between 25 mM and 100 mM. Insome embodiments, the salt concentration is 50 mM. In some embodiments,the salt concentration is less than 10 mM. In some embodiments, the saltconcentration is more than 250 mM.

In some embodiments, the HA compositions of the invention include one ormore antioxidants. Antioxidants are substances capable of inhibitingoxidation by removing free radicals from solution. Antioxidants are wellknown to those of ordinary skill in the art and include materials suchas ascorbic acid, ascorbic acid derivatives (e.g., ascorbylpalmitate,ascorbylstearate, sodium ascorbate, or calcium ascorbate), butylatedhydroxy anisole, buylated hydroxy toluene, alkylgallate, sodiummeta-bisulfite, sodium bisulfite, sodium dithionite, sodiumthioglycollic acid, sodium formaldehyde sulfoxylate, tocopherol andderivatives thereof, (d-alpha tocopherol, d-alpha tocopherol acetate,d-alpha tocopherol succinate, beta tocopherol, delta tocopherol, gammatocopherol, and d-alpha tocopherol polyoxyethylene glycol 1000succinate) monothioglycerol and sodium sulfite. Such materials aretypically added in ranges from 0.01 to 2.0%.

In some embodiments, the HA compositions of the invention include one ormore isotonicity agents. This term is used in the art interchangeablywith iso-osmotic agent, and is known as a compound which can be added toa pharmaceutical preparation to increase the osmotic pressure, such asan osmotic pressure of 0.9% sodium chloride solution, which isiso-osmotic with human extracellular fluids, such as plasma. Preferredisotonicity agents to be used in the compositions of the inventioninclude are sodium chloride, mannitol, sorbitol, lactose, dextrose andglycerol.

In some embodiments, the HA compositions of the invention include one ormore preservatives. Suitable preservatives include but are not limitedto: chlorobutanol (0.3-0.9% w/v), parabens (0.01-5.0%), thimerosal(0.004-0.2%), benzyl alcohol (0.5-5%), phenol (0.1-1.0%), and the like.

In some embodiments, the composition includes one or more componentsthat minimize unwanted side-effects during injection of the composition.

V. Kits and Articles of Manufacture Comprising the HyaluronanCompositions of the Invention

Also within the scope of the present invention are kits comprising theHA compositions of the invention and instructions for use. The term“kit”, as used herein, refers to a packaged product comprisingcomponents with which to administer the HA composition of the inventionfor treatment of a disease or disorder, e.g., joint pain. The kitpreferably comprises a box or container that holds the components of thekit. The box or container is affixed with a label or a Food and DrugAdministration approved protocol. The box or container holds componentsof the invention which are preferably contained within plastic,polyethylene, polypropylene, ethylene, or propylene vessels. The vesselscan be capped-tubes or bottles. The kit can also include instructionsfor administering an HA composition of the invention.

The kit can further contain one more additional reagents and/ormedications, such as non-steroidal anti-inflammatory drugs (NSAIDs) ornutritional supplements, e.g., supplements comprising glucosamine and/orchondroitin sulfates. Non-limiting examples of NSAIDS include aspirin,diflunisal, salsalate, choline magnesium trisalicylate, ibuprofen,dexibuprofen, naproxen, fenoprefen, ketoprofen, dexketoprofen,flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac,etodolac, ketorolac, diclofenac, aceclofenac, nabumetone, piroxicam,meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, melfenamic acid,meclofenamic acid, flufenamic acid, folfenamic acid, Celecoxib,Rofecoxib, Valdecoxib, Parecoxib, Lumiracoxib, Etoricoxib, Firocoxib,nimesulfide or licofelone. Kits typically include a label indicating theintended use of the contents of the kit. The term label includes anywriting, or recorded material supplied on or with the kit, or whichotherwise accompanies the kit.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with a composition of the invention. In oneembodiment, a container filled with a composition of the invention is apre-filled syringe. In a specific embodiment, the compositions of theinvention are formulated in single dose vials as a sterile liquid.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

In one embodiment, a container filled with a composition of theinvention is a pre-filled syringe. Any pre-filled syringe known to oneof skill in the art may be used in combination with a composition of theinvention. Pre-filled syringes that may be used are described in, forexample, but not limited to, PCT Publications WO05032627, WO08094984,WO9945985, WO03077976, U.S. Pat. No. 6,792,743, U.S. Pat. No. 5,607,400,U.S. Pat. No. 5,893,842, U.S. Pat. No. 7,081,107, U.S. Pat. No.7,041,087, U.S. Pat. No. 5,989,227, U.S. Pat. No. 6,807,797, U.S. Pat.No. 6,142,976, U.S. Pat. No. 5,899,889, US Patent PublicationsUS20070161961A1, US20050075611A1, US20070092487A1, US20040267194A1,US20060129108A1. Pre-filled syringes may be made of various materials.In one embodiment a pre-filled syringe is a glass syringe. In anotherembodiment a pre-filled syringe is a plastic syringe. One of skill inthe art understands that the nature and/or quality of the materials usedfor manufacturing the syringe may influence the stability of an HAcomposition stored in the syringe. In one embodiment, a pre-filledsyringe comprises a silicone based lubricant. In one embodiment, apre-filled syringe comprises baked on silicone. In another embodiment, apre-filled syringe is free from silicone based lubricants. One of skillin the art also understands that small amounts of contaminating elementsleaching into the formulation from the syringe barrel, syringe tip cap,plunger or stopper may also influence stability of the composition. Forexample, it is understood that tungsten introduced during themanufacturing process may adversely affect formulation stability. In oneembodiment, a pre-filled syringe may comprise tungsten at a level above500 ppb. In another embodiment, a pre-filled syringe is a low tungstensyringe. In another embodiment, a pre-filled syringe may comprisetungsten at a level between about 500 ppb and about 10 ppb, betweenabout 400 ppb and about 10 ppb, between about 300 ppb and about 10 ppb,between about 200 ppb and about 10 ppb, between about 100 ppb and about10 ppb, between about 50 ppb and about 10 ppb, between about 25 ppb andabout 10 ppb.

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial, pre-filled syringe or other container that ishermetically sealed. In one embodiment, the unit dosage form is providedas a sterile particulate free HA composition that is suitable forparenteral administration, e.g., injection into the knee or axial andappendicular joints.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question, as well as how and how frequently toadminister the pharmaceutical. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, and other monitoring information.

VI. Methods of Treatment Using the Hyaluronan Compositions of theInvention

The present invention also provides methods of treating, reducing orpreventing at least one symptom associated with joint dysfunction. Themethods include administering to a subject in need thereof atherapeutically effective amount of a composition of the invention, suchthat the at least one symptom associated with joint dysfunction istreated, reduced or prevented. In other embodiments, the presentinvention also provides methods for treating or preventingosteoarthritis, the methods including administering to a subject in needthereof a therapeutically effective amount of a composition of theinvention, such that osteoarthritis is treated or prevented. In someembodiments, the present invention also provides methods for improvingjoint function.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status or a pathological condition, e.g., at least one symptomassociated with joint dysfunction, such as osteoarthritis pain. Atreatment or preventive effect is also evident by a failure to worsen orto develop symptoms where they would otherwise be anticipated. As anexample, a favorable change of at least 10% in a measurable parameter ofdisease, and preferably at least 20%, 30%, 40%, 50% or more can beindicative of effective treatment. The term “prevent” or “preventing”,as used herein, comprises, e.g., prevention of re-occurrence of leastone symptom associated with joint dysfunction, e.g., osteoarthritispain, in a subject who has previously experienced the at least onesymptom.

In some embodiments, the subject is a human, a mammal, e.g., a cat, adog, a farm animal (such as a cow, a sheep, a horse, a donkey), or arodent. In a specific embodiment, the subject is a human. In anotherspecific embodiment, the subject is a horse, such as a race horse, or adog.

In some embodiments, the “at least one symptom associated with jointdysfunction” may be caused, e.g., by a pathological condition.Non-limiting examples of such pathological conditions includeosteoarthritis, rheumatoid arthritis, fibromyalgia, infection orinflammation of the joint. The at least one symptom associated withjoint dysfunction may also be caused by a medical procedure, such asarthroscopy, orthoplasty, injury or long immobilization. In someembodiments, the at least one symptom associated with joint dysfunctionis pain or reduced mobility of the joint.

As used herein, the term “reducing at least one symptom” comprisesdiminishing, ameliorating or eliminating at least one symptom associatedwith joint dysfunction, such as pain or reduced mobility. This term alsocomprises reducing the total number of movement-evoked nerve impulses,or reducing the mean number of impulses per movement, in intact or ininflamed joints after administering an HA composition of the invention.This term also comprises reducing the extent of the activation of ionchannels, such as TRPV1 channels, that are involved in the process ofpain transduction in neurons, upon administration of an HA compositionof the invention. Activation of such channels may be measured, e.g., bymeasuring the change in intracellular Ca²⁺ in neurons after anociceptive impulse, or by measuring whole-cell currents in neurons,upon administering an HA composition of the invention. Furthermore, theterm “reducing at least one symptom” also comprises diminishingnociceptive firing of neurons in an inflamed joints upon administrationof an HA composition of the invention. In some embodiments, the HAcompositions of the invention are more effective at reducing at leastone symptom associated with joint dysfunction than other HAcompositions, e.g., other HA compositions that are currentlycommercially available, such as Synvisc®. In some embodiments, HAcompositions of the invention are able to reduce at least one symptomassociated with joint dysfunction by about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95% or about 96%, 97%, 98% or 99%.

In some embodiments, the methods of reducing at least one symptomassociated with joint dysfunction comprise administering to a subject inneed thereof a therapeutically effective amount of a composition of theinvention. The term “therapeutically effective amount”, as used herein,is intended to include an amount of an HA composition of the inventionthat, when administered to a subject in need thereof, is sufficient totreat, prevent or reduce at least one symptom associated with jointdysfunction or is sufficient to treat or prevent osteoarthritis. One ofordinary skill in the art, e.g., a physician, would be able to easilyascertain the amount of HA composition that would be therapeuticallyeffective. In general, a therapeutically effective amount of thecomposition is between about 0.1 to about 500 mg, e.g., about 0.1 mg,about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg,about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg,about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg,about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg,about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg,about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg,about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg,about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg,about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, orabout 500 mg.

In some embodiments, the therapeutically effective amount of an HAcomposition of the invention is sufficient to achieve an effectiveconcentration of HA inside the knee joint, or in other axial orappendicular joints. Accordingly, the effective amount of HA issufficient to achieve an intra-articular HA concentration of greaterthan 3%, e.g., 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%,9.5%, 10%, or greater than 10%.

In carrying out the presently described methods, the compositions of theinvention may be administered by any administration route determined tobe suitable by one of ordinary skill in the art. In one embodiment, thecompositions of the invention are administered by parenteraladministration. In a specific embodiment, the HA compositions of theinvention may be administered by an injection, e.g., by intra-articularinjection. In some embodiments, a treatment regimen may comprise asingle intra-articular injection. In other embodiments, a treatmentregimen may comprise multiple intra-articular injections, e.g., 2, 3, 4,5, 6 or more than 10 injections. One of skill in the art would be ableto determine the appropriate treatment regimen and the timing for the HAcompositions of the invention for each subject.

In some embodiments of the methods provided herein, the compositionswill be administered as a single intra-articular injection or inmultiple, e.g., 2, 3, 4, 5, 6 injections. In general, 2-6 mL ofhyaluronan composition at a concentration of 4% will be administered.However, it should be appreciated that the volumes of compositionadministered may be of larger volume and/or higher concentration. Thus,in some embodiments, the volume of the composition of hyaluronanadministered is at least 0.1 mL, at least 0.5 mL at least 1 mL at least2 mL, at least 3 mL, at least 4 mL, at least 5 mL, at least 6 mL, atleast 7 mL, at least 8 mL, at least 9 mL, at least 10 mL, at least 11mL, at least 12 mL, at least 13 mL, at least 14 mL, at least 15 mL, atleast 16 mL, at least 17 mL, at least 18 mL, at least 19 mL, at least 20mL, at least 30 mL or more. In some embodiments, the composition ofhyaluronan administered is between 1-30 mL, between 2-20 mL, between2-10 mL, between 2-8 ml, between 3-6 mL, or between 4 and 5 mL.

In some embodiments, the volume of an HA composition of the inventionadministered to a subject is sufficient to fill a cavity in thesubject's joint, e.g., the knee, elbow, hip or other appendicular oraxial joint. The volume of the HA composition is also sufficient to coatthe synovium in the subject's joint. In certain embodiments, the volumeof the HA composition of the invention administered to a subject issufficient to prevent dilution of the HA composition by the fluid insidethe joint. For example, the volume of the HA composition of theinvention is sufficient to maintain an HA concentration of 3% orgreater, e.g., 4%, in the subject's joint.

In certain embodiments, the volume of the HA compositions of theinvention administered to a subject in need thereof is sufficient forimproving joint function, e.g., sufficient to reduce joint pain, yet, issufficiently small, such as to prevent build-up of positive atmosphericpressure inside the joint.

In one aspect, the invention provides methods for improving jointfunction (e.g., knee, elbow, hip, shoulder joint, or other axial andappendicular joints). Improving joint function, as used herein, refersboth to the improvement of the mechanical function (e.g., the ability touse the joint such as by, for instance, walking, running and the use ofthe hands), and the ability to reduce unwanted side effects (e.g., pain)associated with joint function. In one aspect, the disclosure provides amethod of reducing pain associated with joint function. However, itshould be appreciated that the improvement in joint function is notlimited to improvement of the mechanical function and reduction of pain.Any improvement in joint function such as a reduction in inflammation orreduction in swelling is encompassed by the current methods.

In one aspect, the invention provides methods for treatingosteoarthritis. Osteoarthritis (OA) is a degenerative joint diseasecharacterized by a group of mechanical abnormalities involvingdegradation of joints including articular cartilage and subchondralbone. Symptoms associated with osteoarthritis include joint pain,tenderness, stiffness, locking. Osteoarthritis can affect any joint inthe body. The most commonly affected joints are hands, feet, spine, hipand knee. Treating osteoarthritis, as used herein, refers both to theimprovement of the mechanical function of the affected joint (e.g., theability to use the joint such as by, for instance, walking, running andthe use of the hands), and the ability to reduce the pain of theaffected joint.

In one aspect, the present invention provides methods for improvingmechanical joint function. In one aspect, the disclosure providesmethods for improving the functionality of a joint affected withosteoarthritis. In one aspect, the methods for improving joint functionand the functionality of a joint affected with osteoarthritis compriseintroducing by injection into a joint a therapeutically effective amountof a composition of the invention, e.g., a composition comprisinghyaluronan characterized by high elasticity. Improvement in jointfunction and improvement in the functionality can be evaluated relativeto the functionality prior to treatment or compared to a subject who isnot receiving the treatment. In some embodiments, improvement infunctionality is measured by reference to a baseline of functionalityexperienced by the subject prior to being treated with the compositionsprovided herein. For example, in one embodiment, the subject mayexperience an improvement in functionality based on KOOS (Knee andOsteoarthritis Outcome Score) function in daily living (See, e.g., Rooset al., J. Orthop. Sports. Phys. Ther., (1998) 28:22-96). In someembodiments of the methods provided herein, the subject may experienceat least a 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 point change frombaseline based on the KOOS function in daily living prior to receivingtreatment. In some embodiments of the methods provided herein, thesubject may experience at least a 5, 10, 15, 20, 25, 30, 35, 40, 45, or50 point change from baseline based on the KOOS function in daily livingcompared to a subject not receiving treatment.

In some embodiments, the subject may experience an improvement infunction measured by WOMAC (Western Ontario and MacMaster UniversitiesOsteoarthritis Index) function subscale (see, e.g., Bellamy et al., Ann.Rheum. Dis., (2005), 64:881-885). In some embodiments of the methodsprovided herein, the subject may experience at least a 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or 100% improvement in function from baselinebased on the WOMAC function subscale prior to receiving treatment. Insome embodiments of the methods provided herein, the subject mayexperience at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%improvement in function from baseline based on the WOMAC functionsubscale compared to a subject not receiving treatment.

In some embodiments, the improvement in joint functionality may beassessed using a questionnaire with the 5-point Likert scale.

In one aspect, the present invention provides a method of reducing painassociated with joint function. In another aspect, the present inventionprovides a method of reducing pain associated with osteoarthritis. Insome embodiments, reduction of pain is measured by reference to abaseline of pain experienced by a subject prior to being treated withone of the compositions provided herein. For example, in someembodiments, the patient experiences a reduction of pain based on thecommonly known KOOS (Knee and Osteoarthritis Outcome Score) painsubscale score which quantifies a subject's experience of pain based ona known range of factors (see, e.g., Roos et al., J. Orthop. Sports.Phys. Ther., (1998) 28:22-96). For example, in some embodiments, thesubject may experience at least a 5, 10, 15, 20, 25, 30, 35, 40, 45, or50 point change from baseline based on the KOOS pain subscale prior totreatment. In some embodiments, the subject may experience at least a 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 point change from baseline basedon the KOOS pain subscale compared to a subject not receiving treatment.

In some embodiments, the subject may experience a reduction of painmeasured by the WOMAC (Western Ontario and MacMaster UniversitiesOsteoarthritis Index) pain subscale (see, e.g., Bellamy et al., Ann.Rheum. Dis., (2005), 64:881-885). For example, in some embodiments, thesubject may experience at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or 100% reduction in pain from baseline based on the WOMAC pain subscaleprior to treatment. In some embodiments, the subject may experience atleast a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in painfrom baseline based on the WOMAC pain subscale compared to a subject notreceiving treatment.

In some embodiments, the reduction of pain in a subject may be assessedusing a questionnaire with the 5-point Likert scale.

A variety of treatment regimes are encompassed by the methods disclosedherein. For instance, a subject may receive a first dose of thehyaluronan compositions disclosed herein followed by additional doses.In some embodiments, a first dose is administered followed by a seconddose at a specific interval. In some embodiments, the second dose isadministered about 30 days, about 60 days, about 90 days, about 120days, about 150 days, about 180 days, about 210 days, about 240 days,about 270 days, about 300 days, about 330 days, or about 360 days afterthe first dose. It should be appreciated that the dose regime may beadjusted based on the improvement in functionality and/or reduction inpain experienced by the subject. In some embodiments, the subject willreceive a dose every month, every two months, every three months, everyfour months, every five months, every six months, every seven months,every eight months, every nine months, every ten months, every elevenmonths, or every twelve months. In some embodiments a dose isadministered as a single or multiple intra-articular injection(s).

In one aspect, the compositions disclosed herein are administereddirectly to the pathological joints. For example, the compositions canbe administered directly to a knee joint, hip joint, finger or thumbjoint, toe joint, ankle joint, wrist joint, shoulder joint, elbow jointor joints of the spine. In some embodiments, the compositions areadministered to a knee joint. In some embodiments, the compositions areadministered to a shoulder joint. In some embodiments, the compositionsare administered to a hip joint. In some embodiments the compositionsare administered to a joint in the leg or arm. In some embodiments thecompositions are administered to a joint in the leg or arm, other than aknee, shoulder or hip joint, or to an axial or appendicular joint. Insome embodiments of any of the methods provided herein, the joint is ajoint of the axial skeleton. In some embodiments of any of the methodsprovided herein, the joint is the temporomandibular or cranial joint.

In one aspect, the compositions are administered by introducing byinjection into a joint a therapeutically effective amount of thecomposition. In some embodiments, the compositions are administered byintra-articular injection. In some embodiments, the compositions areadministered via injection through a syringe and needle. In someembodiments, the compositions are administered topically at the site ofthe joint. In some embodiments, the compositions are administeredperiarticularly.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein by reference.

EXAMPLES

Unless provided otherwise, the hyaluronan used in the compositionsdescribed herein is obtained from animal, human or bacterial sources.Unless provided otherwise, the compositions used herein are inphysiological buffers.

Example 1 Elastic Properties of the HA Compositions of the Invention

The purpose of this experiment was to investigate the elastic propertiesof commercially available hyaluronan (HA) products of HA compositions ofthe invention. HA used in the preparation of was obtained from bacterialcell wall source and had an average molecular weight of 1-2 million. Thecompositions were prepared in physiological saline (8.47 g/L NaCl) or aphosphate buffer solution (8.47 g/L NaCl, 0.047 g/L NaH₂PO₄.H₂O, 0.213g/L Na₂PO₄) with pH of 5.5-7.5. If samples were heat sterilized afterpreparation, the autoclave cycle used was either 121° C. for 15 minutesor 131° C. for 2 minutes with flash cooling of the material immediatelyfollowing the cycle.

Measurements of the elastic properties of HA samples were conducted on aStresstech High Resolution Research Rheometer from Reologica InstrumentsAB, Inc. using RheoExplorer software version 5.0.40.9. A frequency sweeposcillation test was performed and the elastic modulus (G′) wasdetermined. The frequencies of interest range from 0.1 to 10 Hertz (Hzor cycles/sec) which correspond to the degree of movement or stress inthe human knee joint during standing, walking, running and jumping.

Table 1 below shows HA concentration and average molecular weight inselected HA commercial products for viscosupplementation.

TABLE 1 HA Concentrations and Average Molecular Weights for CommercialHA Products Product Manufacturer Average MW of HA Concentration of HASynvisc ® Sanofi 5 million 0.8% (8 mg/mL) Synvisc ® One Sanofi 5 million0.8% (8 mg/mL) Euflexxa ® Ferring Pharmaceuticals 3 million 1% (10mg/mL) Supartz ® Seikagaku Corp. 1 million 1% (10 mg/mL) Gel-One ®Seikagaku Corp. 1 million 1% (10 mg/mL) (before cross-linking) Hyalgan ®Fidia Pharma 0.6 million 1% (10 mg/mL) Orthovisc ® Anika Therapeutics,Inc. 2 million 1.5% (15 mg/mL) Monovisc ® Anika Therapeutics, Inc. 1-2.9million 2.2% (22 mg/mL) Synocrom ® Chroma-Pharma GmbH. 1.6 million 1%(10 mg/mL) Synocrom ® Mini Chroma-Pharma GmbH 1.6 million 1% (10 mg/mL)Synocrom ® Forte Chroma-Pharma GmbH 2.1 million 2% (20 mg/mL) Synocrom ®Forte One Chroma-Pharma GmbH 2.1 million 2% (20 mg/mL) Synolis V-AAnteis 2 million 2% (20 mg/mL)

The elastic properties of selected commercially available HA productsfor viscosupplementation were evaluated by measuring the elastic modulus(G′) over frequencies ranging from 0.1 to 10 Hertz (Hz or cycles/sec).Joints operate at frequencies ranging from 0.1 to 7 Hz, with frequenciesdetermining the force that the joint surfaces experience in standing up,walking, running and jumping. The elastic behavior of HA in thisfrequency range determines the transmission of mechanical forces to bothjoint surfaces in the knee joint. Table 2 below shows the measuredvalues of G′ (in Pascal) at various frequencies in five HA commercialproducts, and FIG. 1, Panel A shows the same data in a graph format.

TABLE 2 G′Values (in Pascal) for HA Commercial Products Frequency (Hz)Orthovisc ® Synvisc ® Euflexxa ® Supartz ® Hyalgan ® 7 188 131 117 30 65 170 125 109 10 12 2 156 120 103 21 2.0 1 117 106 84 10 0.5 0.5 64 8056 2 0.08 0.1 22 49 30 NA NA

Table 3 below shows the measured values of G′ (in Pascal) at variousfrequencies in several inventive compositions, and FIG. 1, Panel B showsthe same data in a graph format.

TABLE 3 G′Values (in Pascal) for Compositions of the Invention HAConcentration Frequency (Hz) 2.0% 3.5% 4.5% 6.5% 8.9% 7 423 1,020 2,1424,898 6,920 5 394 957 2,003 4,623 6,530 2 315 788 1,691 4,029 5,850 1232 604 1,356 3,376 5,010 0.7 193 515 1,189 3,021 4,550 0.5 157 4351,025 2,678 4,110 0.1 54 168 459 1,368 2,290

Table 4 below shows comparison of G′ values for selected HA commercialproducts and selected HA compositions of the invention.

TABLE 4 Comparison of G′values at different frequencies for HAcommercial products and HA compositions of the invention Walking,Running, Jumping, 0.5 Hz 2.5 Hz 5 Hz HA Commercial Products Orthovisc ®64 160 170 Synvisc ® 80 125 125 Euflexxa ® 56 110 109 Supartz ® 2 22 10Hyalgan ® 0.08 3 2 Inventive Compositions HA concentration 3.5% 433 800957 4.5% 1,025 1,700 2,005 6.5% 2,678 4,100 4,600 8.9% 4,110 6,000 6,530

As can be seen from the data, the compositions of the invention arecharacterized by much higher G′ values than any of the tested HAcommercial products. The G′ values of HA compositions reflect theability of HA molecules to act as a coiled molecular shock absorber andbehave like an elastic solid.

Example 2 In Vivo Analgesic Effects of the HA Compositions of theInvention

Thin nerve filaments from the saphenous nerve of adult male Wistar ratswere dissected and placed on a silver wire electrode and nerve impulsesin pain nerve fibers evoked by mechanical stimulation were assessed.Joints were rotated to replicate innocuous movement (within the workingrange of the joint) and noxious movement (exceeding the working range ofthe joint). Rotations that included an innocuous movement followed by anoxious movement, i.e., a movement cycle, were repeated every 5 minutesfor the duration of the experiment. The nerve activity was analyzed byseparately counting the number of nerve impulses during the innocuousand during the noxious components of each individual movement cycle. Thenumbers of nerve impulses evoked by each component (non-noxious andnoxious) were summed to obtain the total number of impulses of themovement cycle at each time point.

The experiment was conducted to test the analgesic effects ofcompositions containing HA of different average molecular weights (from0.2 to 6 million) and increasing concentrations (from 1% to 6%) on jointpain receptors of intact and inflamed joints. In a group of experiments,kaolin-carrageenan was injected intra-articularly into joints to inducejoint inflammation. One hour later, HA solutions of differentconcentrations and average molecular weight or saline control wereinjected intra-articularly, and the time course of the analgesic effectwas determined. The analgesic effect was assessed by measuring impulsesin pain joint nerve fibers during 8 hours following the intra-articularinjection of the test substance.

FIG. 2 summarizes the results. Shown in Panel A is the average number ofimpulses per movement evoked by a complete movement cycle repeated every5 min. The impulses were measured in single fibers of the medianarticular nerve of inflamed joints treated for 24 hours with thecompositions of different HA concentrations and average molecularweights, as indicated in the right inset. It is clear that solutionscontaining HA of an average MW of 1.3-1.6 million or greater and atconcentrations of 4% or greater are most effective in reducing thenumber of nerve impulses evoked by successive movement cycles. Panel Bshows the mean number of impulses per movement as a function of theaverage molecular weight for 4% HA compositions. The results demonstratehigher efficacy of compositions having the average molecular weight of 1million or greater. Panel C shows the mean number of impulses permovement as a function of HA concentration. The results evidence maximaleffects obtained with 4% HA compositions. The effect of Synvisc®injection is marked by the black triangle. Panel D shows the summary ofthe mean effects on movement-evoked activity in inflamed knee joints 24hours after treatment with HA compositions of different concentrationsand average molecular weights. HA2 corresponds to HA of the averagemolecular weight of 1.3 million, provided by Croma-Pharma GmbH. HA1corresponds to the HA composition provided by the Matrix BiologyInstitute.

The results presented in FIG. 2 demonstrate that 4% HA compositions withthe average molecular weight of 1 million are very effective at reducingthe mean number of nerve impulses in the inflamed joints and istherefore effective at reducing pain in the inflamed joints.

Accordingly, pain nerve impulse activity evoked by movement in inflamedjoints is high and remains stable through the recording period.Injection of 4%-6% HA, with average MW of 1.3 million or over had apronounced inhibitory effect on the movement-evoked impulse activity. Asimilar reduction was obtained with 6% HA MW of 1.6 million. Reductionof the concentration of HA maintaining constant the MW or employing 4%HA solution of lower MW reduced the inhibitory effect. Results obtainedwith the commercial HA solution Synvisc®, which contains 0.8% HA with aMW around 6 million has been also represented for comparison. It can besafely concluded that HA of an average MW over 1.3 million exerts apowerful inhibitory effect on movement-evoked joint nociceptor activity.

FIGS. 3 and 4 show that the 4% HA composition affects both the nerveimpulse activity evoked by the innocuous and by the noxious componentsof the movement cycle. FIG. 3 represents the number of nerve impulsescounted during the non-noxious part of the movement in inflamed jointsthat had been injected 24 hour prior with saline, 4% HA or Synvisc®.FIG. 4 shows the data corresponding to the noxious part of the movementcycle. These results confirm the efficacy of the 4% HA composition inreducing the overall responsiveness of joint pain nerve fibers tomovement-evoked pain activity.

FIG. 5 shows long-term effects of the intra-articular injection of 4% HAcomposition on nerve impulse activity. Panel A shows the average numberof impulses per movement recorded at one day, one week, two weeks andthree weeks after the onset inflammation, followed by an intra-articularinjection of saline or 4% HA composition. Panel B shows percentdifference in the mean number of impulse/movement measured after salineinjection and 4% HA, at the same time points as in Panel A.

The inhibitory effect of HA 4% on movement-evoked activity was alsoobserved in intact joints. As is shown in FIG. 6, Panel A, the totalnumber of movement-evoked impulses in intact animals was around 210impulses per cycle as shown by the data of the upper curve (opencircles). When the same recording was made in intact animals 24 hoursafter injection of 4% HA, the mean number of impulses evoked by the samemovement was very low, below 50 impulses per cycle. This is shown inFIG. 6, lower curve (filled circles) and Panel B, showing the averagedata of 7 experiments. This demonstrates that the effects of HA on jointnociceptors are also present in the non-sensitized fibers.

To determine the time point at which the inhibitory effect of HA startsto develop, the activity evoked by joint movement in two intact rats wasrecorded. After performing 6 movements at 10 min intervals, 4% HAcomposition (Elastovisc™) was injected intra-articularly. Movements wererepeated with the same time interval during the following 8 hours. As isshown in FIG. 6, Panel C, in two individual fibers, the movement-evokedactivity augmented gradually during the first 30-60 minutes afterinjection and then started to decline. Control values were recoveredaround 3 hours of the injection and decreased gradually during the next2 to 3 hours, at which point the experiment was interrupted.

FIG. 7 shows that the inhibitory effect of HA on movement-evokedactivity is dependent on the total volume of HA injectedintra-articularly in intact joints. When the injection volume is 25 μl,the curve representing movement evoked impulse activity (black squares)is similar to the one obtained with intra-articular saline (opencircles). With 50 μl (black triangles) and 75 μl (open triangles), themovement-evoked activity in pain fibers was significantly lower than incontrol.

Example 3 In Vitro Effects of HA Compositions of the Invention on IonChannels Involved in Pain Transduction

The rheological properties of extracellular HA determine theeffectiveness of HA in filtering the transmission of mechanical energyto pain nerve endings in the joint, and are affected by the localinflammatory or degenerative processes. In addition, during injury andinflammation, a large variety of locally released chemical mediators acton peripheral nociceptor endings causing sensitization. However, thepossibility exists that the attenuating effect of HA on pain is also dueto a modification of the transduction and excitation mechanismsactivated by injury in peripheral nociceptors. This modification wouldbe associated to the concentration and size of HA molecules.

Transient Receptor Potential Cation Channel Subfamily V Member 1 (TRPV1)is a non-selective cationic channel that plays a major role in thedetection of noxious stimuli and in the sensitizing effects ofinflammatory mediators on nociceptive nerve endings. TRPV1 behaves as anintegrator of noxious endogenous and exogenous chemical and thermalstimuli in polymodal nociceptor terminals. In joints, TRPV1 has beenimplicated in the inflammatory effects caused by mediators releasedduring long-term systemic painful arthritis.

The purpose of this experiment was to investigate the effect of HA onthe activation of TRPV1, the main ion channel involved in paintransduction. Two cell lines were used for the experiments: the cellline SH-SY5Y VR1 that was genetically modified to have an increasedexpression of TRPV1, and HEK293 cells transfected with the TRPV1+EYFPfusion protein to induce TRPV1 channel expression and identify visuallythe cells containing the TRPV1 channels. It is well established thatheat and capsaicin selectively open the TRPV1 channels, allowing theentrance of calcium ([Ca²⁺]) into the cells, which can be measured usingoptical imaging techniques. Accordingly, excitation of the cellscorresponds to a transient increase in specific cell fluorescence thatwas registered and measured quantitatively. Change in intracellularcalcium concentration caused by the opening of TRPV1 channels inresponse to natural noxious stimuli was measured in individual cells.After loading the cells with a fluorescent calcium probe (Fura-2AM), thecells were stimulated four times with short heat pulses and the changein calcium ions was measured. To define a reliable response to repeatedstimuli with heat excluding tachyphylaxis (i.e., reduction of theresponse to repeated stimuli) the characteristics of the response ofTRPV1 cells to repeated stimuli was defined. Rapid heating pulses of thecell surface up to 47-48° C. were used as the noxious stimulus, appliedfor 30 seconds and repeated at 10 minute intervals (see the exampleshown FIG. 8, panel A1). To quantify the effect of the stimulus, thechange in intracellular calcium ([Ca²⁺]_(i)) occurring under controlledconditions in response to an initial heating pulse in each cell wasmeasured, and the response was averaged for a large number of cells. Aprotocol of repeated stimuli at fixed time intervals was establishedthereafter. The 4th pulse was considered the most stable and the percentreduction of its amplitude, in comparison with the amplitude of thefirst stimulus (taken as a baseline reading of 100%), was considered tobe due to the effect of tachyphylaxis. Hence, the response of the cellsto the 4^(th) pulse was expressed in terms of the percent reduction inthe magnitude of the first response when perfused with the controlsolution.

The effects of HA on TRPV1 channels expressed by fluorescent culturedHEK293 cells transfected with the TRPV1+EYFP fusion protein to induceTRPV1 channel expression, and by dissociated dorsal root ganglion (DRG)primary sensory neurons were investigated by measuring the change inintracellular calcium [Ca²⁺]_(i) evoked by repeated noxious stimuli(heat or application of the TRPV1 agonist capsaicin) before and afterperfusion with HA. HA compositions of an average MW of 5.6 million wereused for these studies and were prepared to obtain a final solutioncontaining 400 μg/ml of HA.

The results of the experiment are shown in FIG. 8. Panel A1 showsintracellular calcium rises evoked in HEK-TRPV1-EYFP (+) cells byheating the bathing solution to 48° C., repeated at 10 min intervals.Cytosolic Ca²⁺ increases are represented as the ratio of the emissionfluorescence intensities at 340 and 380 nm (F340/380: fluorescencearbitrary units). Panel A2 shows the same experiment as in A1, but with400 μg/ml HA perfusion started at the end of the 1st heating stimulus.Panel A3 shows the ratio of average amplitude change between responsesevoked by successive heat pulses (indicated in the abscissae axis) incontrol saline solution (CS, black bars) and during perfusion with HA(gray, bars). It is notable that inhibition appears 20 min after theonset of the HA perfusion.

Panels B1-B3 show the results of an experiment utilizing the sameprotocol as in A1-A3 but performed in the DRG adult neurons. Theinhibitory effect of HA becomes evident after 30 minutes of treatment(see 4th vs 1st stimuli).

Panels C1-C3 show intracellular calcium change in a HEK-TRPV1-EYFP (+)cell in response to 100 nM capsaicin and 10 μM Carbachol (Cch), incontrol saline solution (C1) and under exposure to HA initiated 30 minearlier (C2). Panel C3 shows average amplitude of the response tocapsaicin (filled bars) and Carbachol (striped bars) under perfusionwith control saline solution (black) and in the presence of HA (gray).

Panels D1-D2 show intracellular calcium responses of DRG adult sensoryneurons to 100 nM capsaicin and 30 mM KCl during perfusion with saline(D1) and with HA (D2). Panel D3 shows the average amplitude of theintracellular calcium responses to capsaicin (filled bars) or KCl(striped bars) in control saline solution (black) and in presence of HA(gray).

The results in FIG. 8 demonstrate that after a 30 minute exposure to HA,the response of cultured HEK-TRPV1 cells to heat pulses was reduced by77% as compared to the control cells perfused with saline. In theexperiment utilizing an identical protocol, the response of cultured DRGneurons to heat pulses was reduced by 24% as compared to the control.Also, a significantly lower number of HEK-TRPV1 cells (63%) respondedwith an intracellular calcium increase after stimulation with 100 nMcapsaicin in the presence of HA, with the amplitude of the responsebeing 33% lower as compared to the control conditions. Likewise, 68% ofDRG neurons perfused with saline responded to capsaicin, while only 37%of DRG neurons perfused with HA responded, with the amplitude of theresponse reduced to 44% of control. Accordingly, by reducing theexcitability of neurons, HA can effectively reduce pain.

Another cell line, the SH-SY5Y VR1 was used for experiments where theeffect of the concentration of HA on the responses to heat of TRPV1 wasstudied. Table 5 shows the effect of Low- and High MW HA on theintracellular calcium response to heat in SH-SY5Y VR1 cells. Values ofthe response to the 4^(th) heating pulse are expressed as % of theresponse to the first pulse, under perfusion with saline (control) andwith Low MW HA (average MW 470000 Da) or High MW HA (5.2×10⁶ Da) atconcentrations of 200, 400 and 800 μg/ml (n=total number of cellsmeasured in different experiments for each concentration; t-studentstatistics *P<0.05, **0.01<P<0.001, ***P<0.001). As shown in Table 5,when higher concentration HA solutions of low (600000) or high (5.6M) MWwere applied to SH-SY5Y VR1 cells, their response to heat wassignificantly diminished.

TABLE 5 Effect of Low- and High MW HA on the Intracellular CalciumResponse to Heat in SH-SY5Y VR1 cells. Control LMW HMW 51.8 ± 1.6 (n =510) 200 = 45.3 ± 1.6 (n = 472) * 200 = 22.6 ± 1.2 (n = 353)*** 53.7 ±0.9 (n = 1119) 400 = 33.7 ± 0.6 (n = 1025)*** 400 = 17.9 ± 0.8 (n =400)*** 62.2 ± 1.6 (n = 267) 800 = 32.0 ± 0.5 (n = 765)*** 800 = 16.7 ±0.6 (n = 192)***

Example 4 In Vitro Inhibition of Neuronal Excitability by the HACompositions of the Invention

HA-mediated inhibition of capsaicin-evoked stimulation of HEK-TRPV1cells was also investigated by recording whole-cell currents inHEK-293-TRPV1-EGFP(+) cells. Shown in FIG. 9, Panel A are I-Vrelationships of TRPV1 activated with 100 nM capsaicin in salinesolution (top trace, average of n=19) and in the 400 μg/ml HA solution(bottom trace, average of n=18). Voltage ramps were −120 mV to +10 mV,0.2 Hz, with a slope of 0.8 mV/ms mV/s measured at a voltage of +80 mV.FIG. 9, Panel B shows the average current at +80 mV obtained from theI-V curves shown in Panel A. FIG. 9, Panel C shows values of differentparameters measured from the ramps fitted with a function that combinesa linear conductance multiplied by a Boltzmann activation term,I=g×(V−E_(rev))/(1+exp((V_(1/2)−V/S)). FIG. 9, Panel D shows whole-cellcurrents now at −60 mV in response to 1 μM capsaicin, in controlconditions (top trace), and in cells pre-incubated for 30 minutes andcontinuously perfused with HA (bottom trace). FIG. 9, Panel E showsaverage values of peak currents evoked by capsaicin at −60 mV in salineand in the presence of HA.

The results presented in FIG. 9 demonstrate that capsaicin evokesmembrane currents in HEK-TRPV1 cells and in DRG neurons mediated byTRPV1. The maximum amplitude of the current flowing through TRPV1channels and recorded with the patch-clamp technique in response tocapsaicin was reduced by 72% after exposure to HA. This inhibitoryeffect was still present at physiological values of −60 mV of themembrane potential of the cells. It was also confirmed that the voltagegating mechanism and voltage dependence of TRPV1 was not affected by HA,although the conductance (g) was reduced by 47%.

Modulation of TRPV1 single-channel activity by HA was also investigated.The observed decrease in macroscopic currents caused by HA on HEK-TRPV1cells was explored by measuring single-channel currents before and after30-60 min exposure to HA. The single channel activity evoked by 0.25 μMcapsaicin present in the pipette was recorded in cell-attached patchesof HEK293-TRPV1-EFYP (+) cells. The recordings were performed at +60 mV.

FIG. 10, Panel A shows a sample record of TRPV1 single channel activityunder perfusion with saline solution, while Panel B shows samplerecording of single-channel activity from cells incubated in HA andrecorded under perfusion with HA solution. The insets represent singlechannel amplitude probability histograms of patches recorded undersaline (black) and after exposure to HA (gray). FIG. 10, Panel C showsI-V curves obtained in control conditions (squares) and after exposureto HA (circles), while Panel D shows single channel amplitudes obtainedfrom individual patches, represented as squares (control) and circles(HA-treated). Larger symbols correspond to the data from the measuresperformed in the traces shown in Panels A and B. The bars correspond tothe mean values of single channel amplitudes ±s.e.m in each condition;t-test, P=0.73 N.S.

FIG. 10, Panel E shows the probability of the open state for differentpatches. Larger symbols represent the data from the measurementsperformed in the traces shown in Panels A and B. The bars represent meanvalues of single-channel open probability ±s.e.m; t-student, **P=0.002.Notably, long closing states that were very infrequent in controlconditions increased in their frequency after treatment with HA. Nodifferences could be observed in the incidence of open states betweenHA-treated and control conditions.

The results shown in FIG. 10 demonstrate that in the presence of HA,TRPV1 channel activity in response to capsaicin is absent in about 30%of the patches. Moreover, the probability of occurrence of open statesis dramatically reduced in the presence of HA. The single-channel I-Vrelationship and single-channel current amplitude in control conditionsand in the presence of HA were similar. However, HA caused an increasein the number of long-duration events in the closed-time histograms, asis evidenced by the measurements of the distribution of channel open andclosed times. Accordingly, the results suggest that HA maintains thechannel closed for longer times, thus reducing its probability ofopening. The consequence is that nociceptive neurons become lessexcitable by noxious stimuli that open TRPV1 channels in the presence ofHA.

In nociceptive DRG neurons possessing TRPV1 channels the opening ofthese channels by capsaicin causes a decrease in input resistance,depolarization and action potential firing. The firing frequency ofaction potentials evoked by 1 μM capsaicin was measured withpatch-clamps in dissociated DRG neurons that express TRPV1.Specifically, electrophysiological recordings of DRG neurons wereperformed in the cell-attached configuration at −60 mV holding potentialin the presence of 1 μM capsaicin. FIG. 11, Panel A shows a samplerecord of the response to capsaicin in a single DRG neuron perfused withsaline solution. Mean firing frequency of the response was 16spikes/second. FIG. 11, Panel B shows a sample record of capsaicinstimulation in a DRG neuron treated with HA and recorded in the presenceof HA. Mean frequency of the response was 4.6 spikes/second. FIG. 11,Panel C shows a sample record of a DRG neuron treated with HA andrecorded in HA, in which no response to capsaicin was observed butimpulse discharge could be evoked with 60 mM KCl. The inset showselevation in intracellular calcium produced by a heat pulse in thisneuron. Absence of intracellular calcium rise to the same heat pulse intwo different neurons is apparent in the bottom trace.

FIG. 11, Panel D shows mean firing frequency (left) and individual data(right) of DRG neurons under control conditions (left bar, n=8), andafter exposure to HA (right bar, n=9).

The results presented in FIG. 11 demonstrate that in the presence of HA,4 out of 10 neurons that had previously responded to capsaicin did notfire action potentials, despite their intact excitability to otherstimuli. The average firing frequency evoked by capsaicin in control DRGneurons was significantly reduced in DRG neurons incubated with HA.Finally, sensitization of TRPV1 channels of DRG neurons by theinflammatory mediator bradykinin was significantly reduced by HA.Altogether, these results indicate that in DRG neurons, HA selectivelyinhibits the impulse firing evoked by capsaicin, modulating the functionof TRPV1 through direct or indirect interaction with the channel.

Example 5 Direct Inhibition of Nerve Impulse Activity by the HACompositions of the Invention

In another group of experiments, the latency of the first nocifensiveresponse was measured in wild-type (WT) or TRPV1−/− knockout (KO) miceafter subjecting them to the Hot Plate Test. The latency of the firstnocifensive response (licking, biting, lifting, guarding, shaking thehind paw or jumping), is considered a behavioral expression of the acutepain evoked by the noxious heat stimulus acting on the paws. The latencyvalue was measured and compared between control animals (baseline),animals that received in the paw a subcutaneous injection of 10 μl ofsterile saline solution, of 400 μg/ml HA or of hyaluronidase, the enzymedigesting the native hyaluronic acid surrounding the extracellularmatrix around pain nerve terminals.

As shown in FIG. 12, the latency of the nocifensive response to the 52°C. heat was significantly reduced by hyaluronidase 7 days later, whereasHA increased the latency, reflecting a reduced sensitivity to noxiousheat. The maximal analgesic effects of HA injection were seen 48 hoursafter its injection. In an additional group of animals, HA was injecteddays after the hyaluronidase injection, to replace native hyaluronicacid destroyed by the enzyme. In these conditions, latency recoverednormal values. This data suggests that the HA inhibits TRPV1, thusreducing the TRPV1-mediated sensitivity of the nociceptor endings tonoxious stimuli. When the experiments were repeated ingenetically-modified TRPV1^(−/−) mice, none of the differences in thehot-plate test responses between treatments seen in wild type animalswere observed.

To confirm that the inhibition of TRPV1 channels by HA also occurred inthe TRPV1 channels of the pain fibers of the knee joints that areactivated by joint rotation (FIG. 13A), 10 μM capsaicin was injectedintra-arterially in anesthetized rats as a bolus through a catheter intothe saphenous artery close to the knee joint at regular 30 minuteintervals before and after intra-articular injection of saline or HA.The impulse activity evoked by close intra-arterial injection of 10 μMcapsaicin and responses to controlled rotation of the knee joint weremeasured (see FIG. 13.

The bolus injection of capsaicin elicited a discharge of nerve impulsesin a part of the explored filaments (FIG. 13, Panel B). The number ofimpulses evoked by the first injection (control response) was taken as100% and served to express the amplitude of the response to thefollowing injections. The impulse firing frequency was always reducedafter the second or third stimulus, persisting throughout the completeinjection series (tachyphylaxis) decreasing on the average to 88±13(n=7) of the control response in 180 min when intra-articular saline wasinjected (FIG. 13, Panel B, black squares). When 4% HA was injected, thepercent reduction of the discharge was 31±7 (n=6) which represents a 65%of reduction. This inhibitory effect was higher at the end of theexperiment, 270 min after intra-articular injection of HA, when 84% ofactivity reduction was observed (FIG. 13, Panel B, open squares).Collectively, these findings confirm that HA inhibits directly TRPV1channels in pain nerve fibers of the knee joint.

Example 6 Force Requirements for Ejection of HA Through Various NeedleSizes

The pressure required to eject a 4% HA composition from a 3 ml syringewith needles of different diameter (30-18 G) has been measured and isshown in FIG. 14. Force was exerted by one plate of a two-plate balance,acting perpendicularly on the embolus of the syringe. Weights ofincreasing magnitude were added to the contralateral plate. Asdemonstrated by the results shown in FIG. 14, the HA compositions of theinvention can be administered to subjects using needles with diametersof 30-18 G.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

What is claimed is:
 1. A pharmaceutical composition comprisinghyaluronan, wherein: the hyaluronan is present in said composition at aconcentration of greater than about 30 mg/mL; the hyaluronan has anaverage molecular weight of between about 1 and about 2 million; thehyaluronan is not cross-linked and/or is substantially free of chemicalmodifications; and wherein the composition is substantially free ofother pharmaceutically active substances.
 2. The composition of claim 1,wherein the other pharmaceutically active substance is a protein.
 3. Thecomposition of claim 1, wherein the other pharmaceutically activesubstance is a glycosaminoglycan that is different from hyaluronan. 4.The composition of claim 1, wherein the other pharmaceutically activesubstance is hydroxypropyl methyl cellulose.
 5. The composition of claim1, wherein the other pharmaceutically active substance is a localanesthetic.
 6. The composition of claim 5, wherein the topicalanesthetic is lidocaine or bupivacaine.
 7. The composition of claim 1,wherein hyaluronan is present at a concentration of about 40 mg/mL toabout 60 mg/mL.
 8. The composition of claim 1, wherein the compositionfurther comprises a buffer.
 9. The composition of claim 8, wherein thebuffer is phosphate buffered saline (PBS).
 10. The composition of claim1, wherein the composition has an elasticity of at least about 200Pascal when measured at a frequency of 0.5 Hz.
 11. The composition ofclaim 1, wherein the composition has an elasticity of at least about1,000 Pascal when measured at a frequency of 0.5 Hz.
 12. The compositionof claim 1, wherein the composition has an elasticity of at least about2,000 Pascal when measured at a frequency of 0.5 Hz.
 13. The compositionof claim 1, wherein the composition has an elasticity of at least about4,000 Pascal when measured at a frequency of 0.5 Hz.
 14. Apharmaceutical composition comprising hyaluronan, wherein: thehyaluronan is present in said composition at a concentration of about 40mg/mL; the hyaluronan has an average molecular weight of between about 1and about 2 million; the hyaluronan is not cross-linked and/or issubstantially free of chemical modifications; and wherein thecomposition is substantially free of other pharmaceutically activesubstances.
 15. The composition of claim 14, wherein the otherpharmaceutically active substance is a protein.
 16. The composition ofclaim 14, wherein the other pharmaceutically active substance is aglycosaminoglycan different from hyaluronan.
 17. The composition ofclaim 14, wherein the other pharmaceutically active substance ishydroxypropyl methyl cellulose.
 18. The composition of claim 14, whereinthe pharmaceutically active substance is a local anesthetic.
 19. Thecomposition of claim 18, wherein the topical anesthetic is lidocaine orbupivacaine.
 20. The composition of claim 14, wherein the compositionfurther comprises a buffer.
 21. The composition of claim 20, wherein thebuffer is phosphate buffered saline (PBS).
 22. The composition of claim14, wherein the composition has an elasticity of at least about 200Pascal when measured at a frequency of 0.5 Hz.
 23. The composition ofclaim 14, wherein the composition has an elasticity of at least about1,000 Pascal when measured at a frequency of 0.5 Hz.
 24. The compositionof claim 14, wherein the composition has an elasticity of at least about2,000 Pascal when measured at a frequency of 0.5 Hz.
 25. The compositionof claim 14, wherein the composition has an elasticity of at least about4,000 Pascal when measured at a frequency of 0.5 Hz.
 26. The compositionof claim 1 or 14, wherein the composition is sterile.
 27. Thecomposition of claim 26, wherein the composition is suitable forinjection into a subject.
 28. A method of reducing at least one symptomassociated with joint dysfunction, the method comprising administeringto a subject in need thereof a therapeutically effective amount of thecomposition of claim 1, thereby reducing at least one symptom associatedwith joint dysfunction.
 29. The method of claim 28, wherein the jointdysfunction is joint pain or joint dysfunction associated withosteoarthritis, post-arthroscopy, post-orthoplasty, post-injury orprolonged immobilization.
 30. The method of claim 29, wherein the jointdysfunction is joint pain.
 31. The method of claim 28, whereinadministering of the composition causes a reduction in the number ofnerve impulses induced by normal and abnormal movements in anexperimental rat model of osteoarthritis.
 32. The method of claim 31,wherein the reduction is at least two-fold, at least three-fold, atleast four-fold or at least five-fold.
 33. The method of claim 28,wherein administering of the composition causes a reduction in thenumber of nerve impulses induced by normal movements in an experimentalrat model of osteoarthritis.
 34. The method of claim 33, wherein thereduction is at least two-fold, at least three-fold, at least four-fold,at least five-fold, at least six-fold, at least seven-fold or at leasteight-fold.
 35. The method of claim 28, wherein administering of thecomposition causes a reduction in the number of nerve impulses inducedby abnormal movements in an experimental rat model of osteoarthritis.36. The method of claim 35, wherein the reduction is at least two-fold,at least three-fold or at least four-fold.
 37. The method of claims 31,33 and 35, wherein the reduction is greater than the reduction caused byadministering a comparable amount of Synvisc®.
 38. The method of claim28, wherein the composition is administered by intra-articularinjection.
 39. A device comprising the composition of claim 1 or
 14. 40.The device of claim 39, wherein the device is a pre-filled syringe. 41.The device of claim 39, wherein the pre-filled syringe is a 5-mlpre-filled syringe.
 42. The device of claim 40, wherein the pre-filledsyringe is sterilized.
 43. A kit comprising the pre-filled syringe ofclaim 40.